Changes in gene transcription are important in the progression of cancer, in most other human diseases, and in the aging process, as well as in the development of multicellular organisms at all stages. In recent years it has become clear that chromosome topology plays a vital role in gene regulation, as well as other nuclear processes. The major determinant of this topology involves regulatory DNA known as insulators. Tools available in Drosophila make it possible to study mechanisms of insulator action and interaction in detail, in a true in vivo context. This proposal is to study mechanisms of chromatin-based gene regulation involving insulators, including those recently discovered in the well-characterized Drosophila gene even skipped. These studies will address basic questions of how regulatory DNA that mediates chromosome topology carries out its function in 3 dimensions inside the nucleus. They will show how chromosome architecture interfaces with alternative transcriptional states by integrating the topology of insulator pairing with that of enhancer-promoter interactions. A unique contribution of these studies will be to identify an insulator pairing code that can be used to predict and design chromosome architecture and the consequence effects on gene expression. In mammals, insulators are known to be involved in cell-type specific gene regulation and in key developmental decisions, as well as in oncogenesis and inherited human disorders. Therefore, these studies will have direct applications to research on human diseases. The Specific Aims are: Aim 1. Define the molecular basis of insulator pairing specificity and its effects on chromosome architecture and gene expression. Aim 2. Determine the topological and functional consequences of manipulating endogenous insulators within and surrounding the eve locus. Aim 3. Test whether insulator pairing in cis competes with or facilitates insulator pairing in trans, and determine the resulting effects on gene expression.